The present invention relates to the amplification of nucleic acids, i.e. procedures for producing copies of nucleic acid sequences.
As used herein the term xe2x80x9cnucleic acidxe2x80x9d includes protein nucleic acid (PNA) (i.e. nucleic acids in which the bases are linked by a polypeptide backbone) as well as nucleic acids (e.g. DNA and RNA) having a sugar phosphate backbone.
Various nucleic acid amplification techniques are already known, e.g. the Polymerase Chain Reaction (PCR). However many of these techniques (including PCR) suffer from the disadvantage that various cycles of heating and cooling are required for each amplification reaction. Thus, in a typical amplification reaction, the sequence (in single stranded form) to be amplified is treated with an oligonucleotide capable of hybridising to the sequence at a particular location thereof, the treatment being effected at a temperature (and under other conditions, e.g. buffers etc.) at which the hybridisation will occur. In the next step (which may or may not be effected at the same temperature) a polymerase enzyme is used to extend the oligonucleotide primer (using the original sequence as a template) to produce a strand which is complementary to the original strand and which is hybridised thereto. Subsequently the reaction mixture must be heated to denature the complementary strands and then cooled so that the above described procedure (i.e. primer hybridisation, extension, denaturing) may be repeated.
An alternative amplification procedure known as Strand Displacement Amplification has previously been proposed. This procedure may be effected isothermally but does require the construction of a double stranded nucleic acid molecule incorporating hemi-modified restriction site, more particularly a site modified (in one strand) by the incorporation of a thiolyated adenine. The SDA reaction must be conducted in the presence of a chemically modified base to ensure regeneration of the hemi-modified restriction site. This modified base does however become incorporated in the copy strands produced in the reaction and this is a restriction imposed on the procedure.
According to the present invention there is provided a method of amplifying a nucleic acid sequence present in a first strand of a double stranded nucleic acid molecule comprised of complementary first and second strands wherein
said molecule incorporates an unmodified recognition site for a restriction enzyme capable of cutting the first strand at the 5xe2x80x2 end of the sequence therein to be amplified and leaving the 3xe2x80x2-end region of the second strand projecting beyond the cut site in the first strand, and said method comprises
treating said nucleic acid molecule with said enzyme in the presence of a strand displacing polymerase and unmodified nucleotides for incorporation in an extending nucleic acid strand under conditions such that there is or becomes hybridised to said 3xe2x80x2-end region of the second strand a primer sequence complementary thereto whereby said primer sequence is extended direction using the second strand as a template to re-generate the restriction endonuclease cut site and displace the sequence to be amplified.
By unmodified recognition site we mean that the site consists of unmodified A, G, T and/or C bases.
It will be appreciated that the steps of cutting and extension may be repeated as often as necessary to generate the desired amplification.
An important feature of the invention is that the restriction enzyme is capable of providing a 3xe2x80x2-end region of the second strand which projects beyond the cut site in the first strand. The nucleic acid molecule and/or the nature of the restriction enzyme may be such that only the first strand is cut (i.e. nicked). Alternatively both strands may be cut. In either case, the cut in the first strand einerates a fragment (a 3xe2x80x2-upstream fragment) on the 3xe2x80x2 side of the cut in that strand. This fragment may, in certain cases, act as said primer sequence (provided that it remains hybridised to the 3xe2x80x2-end region of the second strand) Alternatively, depending on the length of the fragment and/or the reaction conditions, the fragment may be cleaved from the end region of the second strand to generate a 3xe2x80x2-overhang. It is therefore usually preferred that an oligonucleotide primer (also referred to herein as the FP primer) capable of hybridising to the 3xe2x80x2-end region is additionally incorporated in the reaction to improve the probability of there being a primer sequence hybridised to the 3xe2x80x2-end region of the second strand for effecting the extension/displacement reaction. It will be appreciated that FP primer incorporates all or part of the overhanging sequence produced by the enzyme digestion outlined above.
The manner in which amplification proceeds to effect amplification is described in more detail below but, in brief, the primer sequence is extended in the 5xe2x80x2 to 3xe2x80x2 direction to displace the sequence to be amplified whilst creating a further copy of that sequence (hybridised to the template strand) and regenerating the restriction site. The processes of cutting the double stranded molecule and extension/displacement are repeated to provide for increasing quantities (i.e. amplification) of the sequence to be amplified.
It is preferred that the nucleic acid molecule incorporates two restriction sites of the type described, one each side of the sequence to be amplified. These restriction sites are ideally the same as each other (so that only one restriction enzyme is required) but may be different. The provision of two restriction sites as described allows for extension/displacement reactions to proceed in opposite directions from either end of the molecule. By providing an excess of FP primers in the reactant mix, these primers may hybridize to the 3xe2x80x2-overhangs, produced by the digestion with the restriction enzyme, allowing production of double stranded molecules incorporating a single restriction site. These double stranded molecules participate in further amplification reactions as described more fully below. As described below, such further reactions produce nucleic acid strands which are not able to bind to the FP primers. In an advantageous development of the invention, the reactant mix includes at least one further type of primer (referred to herein as ISOS primers) incorporating the FP sequence and being capable of hybridising to the nucleic acid strands which are themselves not able to bind to the FP primers per se. The ISOS primers result in generation of further double stranded molecules incorporating a restriction site, such molecules being able to participate in amplification reactions. This channelling of otherwise non-hybridisable single strands back into the amplification process leads to an exponential accumulation of product. Where both ISO-S and FP primers are used, the former will generally be employed at a much lower concentration than the latter, generally at least 10 fold less ISO-S primer than FP primer. The ISO-S primers generally are used at a concentration of between 1 fmol/xcexcl to 50 pmol/xcexcl and the FP primers are generally used at a concentration of between 10 fmol/xcexcl to 500 pmol/xcexcl. It should be noted that, under conditions in which the cleavage products of restriction enzyme digestion do not become separated prior to the action of the DNA polymerase, the exponential amplification reaction may be performed by the ISOS primers in the absence of FP primers.
The method of the invention may be a solution phase reaction, and must occur under such conditions (of salt concentration, pH, nucleotide concentration etc.) that both the restriction digestion and polymerase extension reactions can occur, though not necessarily simultaneously. Preferred conditions for the method of the invention to be carried out are in a New England Thermopolymerase buffer at pH 8.8, in the presence of 10-20 mM magnesiwn ions, and a nucleotide final concentration of 0.1-1.0 mM.
Theoretically, the target nucleic acid for the amplification may be present in very small amounts, and hence the amplification will find utility in such areas as clinical diagnostics, to detect either the presence or absence of a sequence or, at a more discriminatory level the occurrence of variations of an underlying sequence. Both DNA and RNA might be used as the target for amplification, the later requiring initial reverse transcription to a DNA intermediate.
The restriction enzyme may for example be TspRI but any other enzyme producing the required 3xe2x80x2-end region in the second strand may be used.
The strand displacing polymerase may for example be 9xc2x0 N polymerase (ex-New England Biolabs), Klenow (exo-) polymerase. Bst polymerase, Vent (exo-) polymerase. or Deep Vent (exo-) polymerase.
The temperature(s) at which the reaction is effected is/are dependent upon the combination of restriction endonuclease and displacing polymerase used in the reaction. Under certain conditions, the restriction endonuclease will effect cutting at the temperature at which the displacing polymerase will effect a copying reaction. Under such conditions, the reaction of the invention may be effected isothermally. It is however also within the scope of the invention that different temperatures are required for cutting and copying so that a two-step thermal cycling reaction may be envisaged. Thermal cycling in this case is not that cycling which is required to perform PCR. In PCR, a strand separation step is an absolute requirement of each cycle of the technique and is usually performed by heating the sample to 95-98xc2x0 C. No such cyclic strand separation is required in the method of the invention where cycling is used to allow primer annealing or to move between the temperature optima of the enzymes used.